The present invention relates in general to the field of biological sample analysis, and more particularly, to an apparatus and method for observing, identifying and quantifying a biological sample through a microscope using the entire spectrum of light, concurrently and in real time.
Without limiting the scope of the invention, its background is described in connection with the observation and analysis of nucleic acid, amino acid, small molecular and/or cellular samples using fluorescence-based microscopy, as an example.
Heretofore, in this field, fluorescence imaging of biologically relevant samples has had an enormous impact on the clinical identification of disease conditions and their prognosis. Likewise, basic research has benefitted from the ready availability of fluorescence-based imaging dyes and image capture systems. In the clinic, developments in immunohistochemistry by fluorescent imaging using fluorescence in situ hybridization (FISH) or comparative genomic hybridization (CGH) have opened new avenues in the identification of chromosomal aberrations from tissue biopsies. In basic research, fluorescence microscopy may be used to detect the presence of markers using techniques such as, e.g., fluorescence or enzymatic labeling, to detect the presence or absence of binding to a component of the sample which has been immobilized.
One such fluorescence-based system is known as Spectral Karyotyping (SKY), which uses Fourier Transform Imaging Spectroscopy Microscopy. In SKY microscopy a continuum of spectra are taken, but require a filter set that is specific for the dye used. The hyperspectral image produced and deconvoluted also requires the use of dyes having known emission spectra and filters adapted therefor. The SKY system deconvolutes images using a two-step process. First, an image is acquired after passage through dye-specific filters into a Sagnac Interferometer followed by inverse-Fourier transform calculations. The interferometry, however, depends on the shape of the bandpass filter and is therefore limited to specific dye-filter sets that exclude photons outside the bandfilter are used. The effect of excluding photons close to the filter wavelength is that sensitivity is sacrificed, but is necessary, due to the intrinsic limits of the Sagnac interferometer. Furthermore, the SKY system fails to account for variation in the quality of filters. The lack of dimensional stability, due to susceptibility in image registration and difficulties in lateral coherence, also requires that the user of the SKY system continuously adjust and monitor the system optics.
It has been found, however, that present apparatus and methods fail to meet the demand for a low cost, efficient, customizable imaging microscope that is capable of overlapping, concurrent data acquisition and analysis over the entire spectrum of light. A problem found in alternative systems is that they are only capable of imaging a limited number of dyes with high quality filters. Another problem with available systems is that constant monitoring and adjustments in the system optics is required, increasing the mechanical complexity of the system.
Further, current systems can not make simultaneous measurements at different wavelengths and thus cannot measure multiple parameters simultaneously. Present systems can not accurately correlate parameters (or measurement conditions) that are changing with time (e.g., due to bleaching), thereby losing sensitivity while an area of interest is being selected and each wavelength is measured.
The hyperspectral imaging microscope of the present invention is designed to greatly enhance the signal, spectral range and sensitivity of microscopic imaging in order to aid, e.g., the clinical technician to evaluate karyotypes for cancer evaluation. In the clinical setting, for example, the microscope disclosed herein allows the cytogeneticist to evaluate a large number of potential genetic abnormalities simultaneously and with high sensitivity.
The hyperspectral imaging microscope (HIM) disclosed herein may record the entire emission spectra of a sample. The microscope of the present invention may be used with or without a set of unique marker specific for detection of chromosomal abnormalities. In fact, the microscope can be used without any dye marking of the sample and be used using plain light. The microscope can be used as part of a Hyperspectral Image Cytogenetics (HIC) system, in which the protocols and the probe set disclosed herein enable the clinical and research cytogeneticist to quickly and accurately gauge chromosomal rearrangements, deletions and duplications with greater accuracy than previously possible using optical microscopy.
The microscope may be constructed using an imaging spectrograph, a cooled high-resolution CCD camera and a raster system. When a sample is fluorescently tagged, the microscope is capable of analyzing samples hybridized with a large set of distinctly tagged probes. Because the entire wavelength spectra may be taken simultaneously, the microscope is able to resolve, spectrally, a large number of emission spectra, whether dyes are used or not. When using dyes with a broad emission spectra, the microscope is capable of capturing the entire broad spectra. The microscope, however, is also able to distinguish close peak spectral spacing, as well as, spectra of compressed samples where dye positions overlay spatially or are very close.
The sensitivity of the system is also improved because the entire spectra of a fluorochrome may be captured. By means of comparison, a filter based epi-fluorescence microscope passes only a narrow passband window of light. Additional benefits of the microscope disclosed herein include, single scan for all wavelengths, no dye specific hardware (emission filters), extendable to near IR, may be automated, and may be used with current FISH and CGH methods. Filters, however, may be included for specific applications.
The present invention is an apparatus for imaging samples that includes a microscope positioned to hold a sample, generally disposed on a slide, in the path of light. A light dispersive element, such as an imaging spectrograph, is positioned to disperse the light emitted from the sample into a light spectrum. Following the interaction of the light line with the sample, a camera detects the light spectrum produced by the light dispersive element to determine the components of light emitted by the sample. The microscope may be, e.g., an epi-fluorescence or an inverted microscope. The camera may be, e.g., a charge coupled device (CCD) camera. A light amplification device, such as a micro-channel plate amplifier, may be placed between the light dispersive element and the camera to improve the sensitivity of this system.
Light sources for use with the present invention may include the light generally associated with the microscope or lasers, lamps, or combinations thereof. Examples of lasers that may be used include: argon ion lasers, diode-pumped solid state lasers, pulsed nitrogen dye lasers, helium-neon lasers or a combination thereof. Lamps for use with the invention include: white light lamps, broadband ultraviolet lamps, mercury lamps, zenon lamps, or combinations thereof. When the sample are stained, the light source may be selected depending on the dye or dyes used to stain the samples on the slide.
In one embodiment of the present invention, the light may be further defined as including a continuum or spectrum of light. The light dispersive element for use with the present invention may be an imaging spectrograph. Also, an astigmatism correcting lens may be positioned between the microscope and the imaging spectrograph. The microscope may include a linear motion drive slide mount, or a linear motion slide mount attached to a light reflective element, such as a collimator or a total internal reflection mirror. The hyperspectral imaging microscope may further include a data acquisition system connected to the camera, wherein the data acquisition system stores and correlates results based on the input acquired by the camera.
The camera of the present invention may be used to detect changes in absorbance of light following the interaction of light with a sample. The camera may, alternatively, detect the reflection of light following the interaction of light with the sample on all or a portion of a slide, or the emission of a fluorochrome.
The present invention also includes a method of scanning samples on a slide in multiple wavelengths including the steps of, generating one or more wavelengths of light, illuminating at least one sample on a microscope with the light, splitting the light into a two dimensional array of light and detecting the two dimensional array of light.
Samples for detection using the apparatus and method of the present invention include biological and materials sciences samples. By material sciences it is meant that samples are primarily of inorganic origin, e.g., materials used for semiconductor manufacture. An example of a semiconductor sample is a dye cut or punch for auger analysis. The hyperspectral imaging microscope may also be used for the study of environmental samples, e.g., high definition analysis of contaminated deposits.
When using the present invention to observe and quantitate biological samples, the samples may be completely unstained, or may be stained using, e.g., a fluorescent dye. The samples may be, e.g., intact or fixed cell, cells grown on slides or histological samples. Techniques used to stain the samples may be, e.g., stained using fluorescence in situ hybridization or comparative genomic hybridization techniques. Alternatively, the biological samples may be tagged antibodies or other proteins (e.g., ligands for cell surface receptors), ion sensitive dyes, intercalating dyes, nucleic acid stains, and the like. The hyperspectral imaging microscope may be used for time-dependent studies that require measurements at several timepoints. It may also be used to study arrays that are used for sequencing, such as, re-sequencing matrices.
The apparatus and method disclosed herein may also be used in clinical settings for fluorescence in situ hybridization (FISH) of cancer cell cytogenetics to establish a molecular basis for diagnosis (single copy probes directed at oncogenes) and the evaluation of complex translocations (whole chromosome painting probes). The apparatus and method may also be used to predict the accelerated phase of disease (centromeric probes on interphase nuclei), monitoring engraftment after bone marrow transplantation (chromosome X and Y probes) and even for the evaluation of marker chromosomes (centromeric or painting probes). Probes for use with the present invention include, but are not limited to, any dyes or molecules that affect light dispersion, emission, reflection, absorbance, emission shifts, energy transfer and the like.